Presentation

1. UNIVERSITY OF NIGERIA, NSUKKA
DEPARTMENT OF MICROBIOLOGY
MSc RESEARCH PROPOSAL
Topic:
EVALUATION OF ROOF-HARVESTED RAINWATER
AND TANKER-SUPPLIED BOREHOLE WATER IN NSUKKA
FOR FAECAL INDICATORS AND BACTERIAL PATHOGENS

2. PRESENTATION OUTLINE
 INTRODUCTION
 AIM & OBJECTIVES OF STUDY
 RESEARCH METHODOLOGY
 TIMELINE
 EXPECTED OUTCOMES

3. INTRODUCTION
 The demand for potable water supply is on the increase due to
the increasing population growth, particularly in urban areas,
along with the upsurge in industrial output and commerce
(Ahmed et al.,2011).
 Potable water is in short supply. The global crisis of potable
water shortage has sent man on the lookout for new water
resources and even more intensively (Achadu et al., 2013).
 The poor economic situation of developing countries has
limited man from having a personal borehole for his household,
therefore sometimes rely on borehole water supplied by
tankers (Khan et al., 1997).

4.  Roof-harvested rainwater (RHRW) has been considered an
effective alternative water source for drinking and various
non-potable uses in a number of countries throughout the
world (Ahmed et al.,2011).
 However, if a person chooses to use rainwater for drinking
or any other purpose, (Ahmed et al., 2012) then that
person is responsible for ensuring that the quality of the
water is sufficient for its intended use.
 Research in this area will help to provide the knowledge
and guidance to ensure that.

5.  The quality of roof-harvested rain water is monitored using
faecal indicators such as Escherichia coli, fecal coliforms,
total coliforms and Enteroccoci (Lye et al., 2002).
 These are microorganisms that have commonly been found
in the guts of humans and other warm blooded animals
(Ahmed et al., 2012).
 Investigations carried out in different parts of the world
show the prevalence of faecal indicators, zoonotic bacterial
(Campylobacter spp., Salmonella spp. etc) and protozoan
(Cryptosporidium parvum, Giardia lamblia, etc) pathogens
in water samples from rainwater tanks.
 Some of these pathogens are becoming highly resistant to
antibiotics (Ahmed et al, 2010).

6. STATEMENT OF THE PROBLEM
 In developing countries like Nigeria, low economic growth and
the effects of climate change are causing difficulties in getting
water from other suitable sources such as boreholes.
 In addition to bore-hole water, many in Nsukka rely heavily on
harvesting rain water as a more economical alternative (Khan
et al., 2012).
 Despite a high level of dependence on rainwater for drinking
and domestic uses in Nsukka, Southeast, Nigeria, little is done
to monitor the quality of roof-harvested rainwater stored in
tanks and reservoirs.
 There are potential public health risks associated with
microbial pathogens (Water Aid, 2013).

7. AIM OF STUDY
The study is aimed at
Evaluating microbiological quality of
tanker-supplied borehole water and roof-
harvested rain water, and the potential
public health risks associated with their
use in Nsukka.

8. SPECIFIC OBJECTIVES
 The specific objectives are to:
1. Determine the pH and temperature of the tanker-supplied
borehole water and roof-harvested rainwater samples
2. Detect and quantify indicator bacteria in tanker-supplied
borehole water and roof-harvested rainwater samples
3. Isolate and characterize bacterial pathogens from the samples
4. Determine the antibiotic susceptibility of isolates
5. Quantify the health risks attendant to the use of contaminated
tanker-supplied borehole water and roof-harvested rainwater

9. METHODOLOGY
STUDY AREA
The university town of Nsukka is a Local Government Area located in
South-East Nigeria in Enugu State with latitude and longitude of
6.8429° N, 7.3733° E respectively (Maps-streetview, 2011).
Nsukka LGA has an area of 1,810 km² and a population of 309,633 at the
2006 census.
Only the University and a few other households in the town have access
to piped-water.
Most people in the town depend on two major water sources: borehole
water supplied by water tankers in the dry season and rainwater
harvested from roofs during the rainy season.
Both tanker-supplied and roof-harvested waters are stored in metal or
plastic tanks.

10. Sample Collection and Water quality
Determination
SAMPLE COLLECTION
A total of 40 water storage tanks will be surveyed twice, once in each
of the dry and rainy periods, giving a total of 80 water samples.
Samples will be collected from eight different selected storage tanks
from each of five selected locations in Nsukka, including Onu-iyi,
Obukpa, Orba, Odenigbo and Barracks.
All samples will be collected in triplicates, following standard
methods.
WATER QUALITY DETERMINATION
 Water quality will be determined using the membrane filtration
method (Brian and Catalina, 2015).

11.  Dilution: Water samples will be diluted serially (10-fold )
will be filtered through a 0.45 µm membrane filter coupled
to a vacuum pump.
 mEndo agar LES, mFC agar and Chromocult Coliform Agar
will be used for detection and enumeration of total
coliforms, fecal coliforms and E. coli and other coliforms
respectively
 After incubation at 37 °C for 24 h, all typical colonies will
be counted
 The standard formula;
colony count (CFUs) on an agar plate
total dilution of tube X volume plated

12. Colonies on membranes incubated on Chromocult
coliform agar (CCA) will be purified on CCA and
designated (based on typical characteristics) as
presumptive E. coli (violet colonies) and non - E. coli
coliform (salmon-red).
All isolates will be subjected to Gram-staining and
standard biochemical tests: Catalase test, Indole test,
Methyl red test citrate test and Voges-proskauer.
E. coli strain (NCTC 13353) and Enterobacter aerogenes
(NCTC 10006) will be used as controls.
Isolation and Preliminary
Identification of Coliforms

13. Antibiotic susceptibility tests will be performed on all
confirmed E. coli isolates following the Kirby-Bauer disc
diffusion method (Bauer et al., 1966) and the evaluation
methods of the Clinical and Laboratory Standards Institute
(CLSI, 2005).
E. coli strain (NCTC 13353) will be used as control strains.
Zones showing complete inhibition around the discs will be
measured and the diameters of the zones will be recorded
to the nearest millimetres.
Antimicrobial Susceptibility Testing

14.  Extraction of Bacterial DNA:
Genomic DNA will be extracted from a pure culture of
each isolate grown overnight on nutrient agar at 37°C, as
described by Vankerckhoven et al., (2004).
Briefly, one loop full of bacterial cells will be suspended
in 1ml of sterile distilled water. The bacterial suspension
will then be heated for 5min at 100oC, cooled in ice and
centrifuged at 10,000 rpm for 10 min to remove the
debris. The supernatant will be used for PCR.
Characterization of Bacterial Pathogens
Using Polymerase Chain Reaction (PCR)

15. Polymerase Chain Reactions:
 Multiplex PCR procedures as described by Guion et al. (2008) will be used to
identify 8 virulence genes of the six classes of diarhoeagenic E. coli and
uropathogenic E. coli.
 The oligonucleotide primers to be used, the target genes and expected
amplification products are listed in Table 1. Positive and negative controls for
the PCR reaction will also be included.
 The positive control will contain a mixture of the DNA of pathogenic and
commensal strains. The negative control will contain PCR grade water. Isolates
lacking the genes will be considered non-pathogenic. After amplification, 6 μl
of each reaction mixture will be subjected to electrophoresis on a 0.8%
agarose gel in TBE (Trisborate-EDTA) buffer.
 The reactions will be subjected to an initial activation step at 95°C for 15 min,
followed by 35 cycles consisting of denaturing at 94°C for 45 s, annealing at
55°C for 45 s, extension at 68°C for 2 min and final elongation at 72°C for 5 min
(Omar and Barnard, 2010).

16. Organism/
Pathotype
Gene (s) Primer
name
Primers Sequence (5'—3') Amplicon
size (bp)
References
Escherichia
coli
uidA EC-F
EC-R
AAAACGGCAAGAAAAAGCAG
ACGCGTGGTTACAGTCTTGCG
147 Bej et al. 1991
EPEC eaeA EP-F
EP-R
TCA ATG CAG TTC CGT TAT CAG TT
GTA AAG TCC GTT ACC CCA ACC TG
482 Vidal et al. 2005
ETEC lt
stII
LT-F
LT-R
ST-F
ST-R
GCA CAC GGA GCT CCT CAG TC
TCC TTC ATC CTT TCA ATG GCT TT
AAA GGA GAG CTT CGT CAC ATT TT
AAT GTC CGT CTT GCG TTA GGA C
218
129
Stacy-Philips et al.
1995
EHEC stx1
stx2
TX1-F
TX1-R
TX2-F
TX2-R
CAGTTAATGTGGTGGCGAAGG
CACCAGACAATGTAACCGCTG
ATCCTATTCCCGGGAGTTTACG
GCGTCATCGTATACACAGGAGC
348
584
Cebula et al. 1995
EAEC aafII EA-F
EA-R
CAC AGG CAA CTG AAA TAA GTC TGG
ATT CCC ATG ATG TCA AGC ACT TC
378 Vidal et al. 2005
DAEC daaE DA-F
DA-R
GAA CGT TGG TTA ATG TGG GGT AA
TAT TCA CCG GTC GGT TAT CAG T
542 Vidal et al. 2005
EIEC ipaH EI-F
EI-R
CTC GGC ACG TTT TAA TAG TCT GG
GTG GAG AGC TGA AGT TTC TCT GC
933 Vidal et al. 2005
UPEC hlyA UP-F
UP-R
TGT TGA AAG ATC AGT CCT CA
CTG CGT AGA TAT TGG CTG AG
500 Maynard et al.
2004
Table 1: Primers for Characterization of Escherichia coli Pathotypes

17.  Risk analysis
The Quantitative microbial risk assessment (QMRA) will be
done using both the exponential and b-Poisson models
(Haas et al. 1999). The QMRA framework consists of four
steps (Chigor et al. 2014; Shrestha et al. 2014). :
 hazard identification,
 exposure assessment,
 dose–response relationship, and
 risk estimation
 Statistical Analysis:
Data will be analysed to determine statistical significance using
analysis of variance (ANOVA). Results with P values of <0.05
will be considered to be significant.

18. TIMELINE/RESEARCH PLAN
S/N What to do When
1 Literature review April 2016 – August 2016
2 Mapping and survey of sampling sites/areas March 2016
3 Preliminary studies/sampling Sep – Dec 2016
4 Presentation of project proposal May 2017
5 Sample collection, water quality analysis and
isolation of bacteria pathogens
February – June 2017
6 Biochemical characterisation of isolates April and June 2017
7 Antibiotic susceptibility testing June 2017
8 Submission of review article manuscript June 2017
9 Molecular characterization of isolates July 2017
10 Statistical analysis of research data August 2017
11 Presentation of research findings to the
Department
August 2017
12 Submission of draft MSc Thesis August 2017
13 Submission of 1st manuscript for research article September 2017

19. Expected Outcomes
It is expected that at the end of this research, the findings
 Could serve as a baseline for proper water quality control
measures in Nsukka.
 Will encourage researchers to go into similar researches
in other places in Nigeria (especially rural areas) where
safety of potable water has been greatly ignored.
 Will motivate individuals to always ensure that the
quality of their water is good for its intended use
regardless of the source.
 Will result in the publication of at least two impact-factor
journal articles.